Prolongation of survival of fully allogeneic cardiac grafts and generation of regulatory cells by a histamine receptor 2 antagonist

BACKGROUND: The effects of histamine on immunologic responses via the histamine receptor 2 (HR2) have been studied, but few investigations explored the immunomodulatory role of histamine in vivo. We examined whether the HR2 antagonist ranitidine affects the alloimmune response in a murine model of cardiac transplantation. METHODS: CBA (H-2k) recipients were given no treatment or one intravenous injection of ranitidine on the day of transplantation of a heart from C57BL/10 (H-2b) donors. Survival of the allografts was recorded. The effect of the ranitidine treatment on cell proliferation and cytokine production was assessed by mixed leukocyte culture and enzyme-linked immunosorbent assays. An adoptive transfer study was conducted to determine whether regulatory cells were generated. The effect on graft survival of adding FK506 to the ranitidine treatment was also examined. RESULTS: CBA recipients given ranitidine (60 mg/kg) had prolonged graft survival (median survival time [MST], 87 days). Ranitidine treatment also suppressed the proliferation of splenocytes and production of interleukin (IL)-2 and up-regulated IL-10 production. Adoptive transfer of splenocytes and CD4 cells from ranitidine-treated allograft recipients induced significant prolongation of allograft survival in naive secondary recipients (MST, 71 and >100 days, respectively). CBA recipients given both ranitidine and FK506 (0.1 mg/kg/day for 14 days) had indefinite survival of cardiac allografts (MST, >100 days). CBA recipients treated with FK506 alone rejected the allografts (MST, 27 days). CONCLUSION: In our model, ranitidine treatment induced significantly prolonged survival of fully allogeneic cardiac grafts, generated CD4 regulatory cells, and indefinite survival when combined with FK506 (0.1 mg/kg/day).     

Role of the CTLA4 pathway in hyporesponsiveness induced by intratracheal delivery of alloantigen

BACKGROUND: The authors previously reported that intratracheal delivery (ITD) of donor alloantigen induced donor-specific hyporesponsiveness to C57BL/10 cardiac allografts in CBA recipients and that blockade of the B7 pathways abrogated that hyporesponsiveness. In this study, the authors used a CD28-deficient model to evaluate which signal, either through CD28 or cytotoxic T-lymphocyte-associated antigen (CTLA4), is involved in the induction of hyporesponsiveness. METHODS: Seven days before transplantation of hearts from C3H/HeJ (H2k) mice into C57BL/6 (H2b) or CD28-deficient (C57BL/6 background) mice, the transplant recipients were given ITD of donor splenocytes (1 x 10(7)), alone or in combination with human CTLA4-immunoglobulin (Ig) (200 microg). RESULTS: ITD of C3H splenocytes induced donor-specific hyporesponsiveness to C3H cardiac grafts in C57BL/6 recipients (graft median survival time [MST], 40 days). Administration of CTLA4-Ig concurrently with ITD abrogated the prolonged allograft survival (MST, 12 days). Interestingly, ITD of C3H splenocytes induced prolonged survival of C3H allografts in CD28-deficient recipients (MST, 55 days). Furthermore, administration of CTLA4-Ig combined with ITD of C3H splenocytes abrogated the prolonged survival of C3H allografts in CD28-deficient recipients (MST, 7 days), whereas recipients given isotype-control antibody in combination with ITD of splenocytes had prolonged survival of C3H allografts (MST, 58 days). CONCLUSIONS: Taken together, the authors' findings indicate that a signal through CTLA4, rather than through CD28, plays an important role in the induction of hyporesponsiveness by ITD of alloantigen in this model.     

Administration of donor splenocytes via the respiratory tract generates CD8α+ regulatory dendritic cells and induces hyporesponsiveness to fully allogeneic cardiac grafts

BackgroundWe previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory T cells (Tregs) in mice. In this study, we examined the role of splenic dendritic cells (DCs) in the ITD model.MethodsCBA mice were treated with ITD from C57BL/10 splenocytes and 7 days later received transplantation of C57BL/10 hearts. In adoptive transfer studies, splenic DCs from ITD-treated mice were transferred into naïve CBA recipients that received C57BL/10 hearts immediately after the transfer. In addition, to determine the role of splenic DCs isolated from ITD-treated mice, the cells were incubated under stimulation with lipopolysaccharide (LPS).ResultsITD-treated CBA recipients had markedly prolonged allograft survival (median survival time [MST], 67 days) while naïve recipients rejected allografts acutely (MST, 8 days). In adoptive transfer studies, CBA recipients of the transfer of splenic DCs from ITD-treated mice had prolonged allograft survival (MST, 85 days), while CBA recipients of the transfer of splenic DCs from naïve mice did not have prolonged allograft survival (MST, 8 days). In another transfer study, CBA recipients of the transfer of splenic CD8α⁺ DCs from ITD-treated mice had prolonged allograft survival (MST, 79 days), while those receiving splenic CD8α⁻ DCs from ITD-treated mice did not have prolonged allograft survival (MST, 8 days). In vitro studies showed that ITD-treated splenic DCs produced more IL-10 and less IL-12 than naïve splenic DCs under stimulation with LPS.ConclusionsITD pretreatment induces regulatory DCs, which produce high amounts of IL-10 resulting in the prolongation of graft survival in our model.     

Induction of indefinite survival of fully allogeneic cardiac grafts and generation of regulatory cells by intratracheal delivery of alloantigens under blockade of the CD40 pathway

BACKGROUND: The authors previously showed that intratracheal delivery (ITD) of donor splenocytes induced prolonged survival of fully allogeneic cardiac grafts in mice. In this study, this treatment protocol was combined with blockade of the CD40 pathway in an attempt to induce operational tolerance. METHODS: CBA mice were given donor splenocytes (1x107) or Kb peptide (100 microg) by ITD with or without antibody specific for mouse CD40 ligand (MR1, 200 microg) 7 days before transplantation of a C57BL/10 heart. Also, splenocyte (5 x 107) from primary recipient CBA mice given ITD of donor splenocytes or Kb peptide plus MR1 were adoptively transferred into naive CBA secondary recipients 7 days after the pretreatment and C57BL/10 hearts were transplanted into those recipients the same day. RESULTS: ITD of donor splenocytes and Kb peptide induced prolonged survival of cardiac grafts (median survival time [MST], 74 and 56 days, respectively), whereas naive control mice and mice pretreated with syngeneic splenocytes had acute graft rejection (MST in both groups, 7 days). When MR1 was included, all grafts survived indefinitely (>200 days), but mice pretreated with MR1 alone had graft rejection (MST, 54 days). Mice bearing cardiac grafts had acceptance of skin grafts from C57BL/10 but not BALB/c mice, demonstrating that operational tolerance was induced. Secondary recipients given adoptive transfer of splenocytes from primary recipients of the combined treatment had acceptance of C57BL/10 grafts, suggesting that regulatory cells were generated within 7 days of pretreatment. CONCLUSIONS: ITD of donor splenocytes or Kb peptide under blockade of the CD40 pathway induced operational tolerance and generated regulatory cells.     

Interleukin-10 but not transforming growth factor-beta is essential for generation and suppressor function of regulatory cells induced by intratracheal delivery of alloantigen

BACKGROUND: We previously reported that intratracheal delivery of alloantigen-induced regulatory cells in mouse heart-transplantation model. Here, we investigated roles of interleukin (IL)-10 and transforming growth factor (TGF)-beta in induction and effector phases of the regulatory cells. METHODS: CBA mice were pretreated with intratracheal delivery of C57BL/10 splenocytes and administration of neutralizing anti-IL-10 or anti-TGF-beta monoclonal antibody (mAb). Seven days after the pretreatment, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes from the pretreated mice and underwent heart grafting from C57BL/10 mice. To determine roles of these cytokines in the effector phase of the regulatory cells, anti-IL-10 or anti-TGF-beta mAb was administered weekly into the secondary recipients after the adoptive transfer. RESULTS: Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST] 68 days) as compared with adoptive transfer from untreated CBA mice (MST 12 days). In the induction phase, anti-IL-10 mAb abrogated development of the regulatory cells that afforded prolonged allograft survival in the secondary recipients (MST 20 days), whereas anti-TGF-beta mAb did not abrogate it (MST 88 days). In the effector phase, anti-IL-10 mAb abrogated prolonged allograft survival afforded by adoptive transfer of the regulatory cells in the secondary recipients (MST 27 days), whereas anti-TGF-beta mAb did not abrogate suppressor function of the regulatory cells (MST 53 days). CONCLUSION: IL-10 but not TGF-beta was required for generation and suppressor function of the regulatory cells induced by intratracheal delivery of alloantigen.     

Programmed death-1-programmed death-L1 interaction is essential for induction of regulatory cells by intratracheal delivery of alloantigen

BACKGROUND: Programmed death (PD)-1 has been implicated in peripheral tolerance. The authors investigated the roles of PD-1 and its ligands, PD-L1 and PD-L2, in the induction of regulatory cells by intratracheal delivery of alloantigen. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of C57BL/10 (H-2b) splenocytes and administration of monoclonal antibody (mAb) specific for PD-1, PD-L1, or PD-L2. Seven days later, C57BL/10 hearts were transplanted into the pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes from the pretreated CBA mice and transplantation of C57BL/10 heart. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 7 days). Pretreatment with intratracheal delivery of C57BL/10 splenocytes prolonged graft survival significantly (MST, 65 days). Administration of control immunoglobulin (Ig) G or anti-PD-L2 mAb did not significantly affect the prolongation (MST, 72 and 68 days, respectively). In contrast, anti-PD-1 or anti-PD-L1 mAb abrogated the prolongation (MST, 18 and 17 days, respectively). Adoptive transfer from mice pretreated with intratracheal delivery of alloantigen plus control IgG or anti-PD-L2 mAb prolonged survival of C57BL/10 grafts in secondary CBA recipients (MST, 72 and 56 days, respectively). However, concurrent administration of anti-PD-1 or anti-PD-L1 mAb abrogated prolonged survival after the adoptive transfer (MST, 14 and 20 days, respectively). CONCLUSIONS: PD-1-PD-L1 interaction was essential for induction of regulatory cells by intratracheal delivery of alloantigen.     

CD27/CD70, CD134/CD134 ligand, and CD30/CD153 pathways are independently essential for generation of regulatory cells after intratracheal delivery of alloantigen

BACKGROUND: We investigated whether blockade of tumor necrosis factor receptor-ligand pathways could generate regulatory cells induced by intratracheal delivery of alloantigen. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of splenocytes (1x10(7)) from C57BL/10 (H-2b) mice and intraperitoneal administration of monoclonal antibody (mAb) specific for CD70, CD134 ligand (CD134L), CD153, or CD137L. Seven days later, C57BL/10 hearts were transplanted into pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes (5x10(7)) from pretreated CBA mice and transplantation of a C57BL/10 heart on the same day. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST] 12 days). Pretreatment with intratracheal delivery of C57BL/10 donor splenocytes prolonged graft survival significantly (MST 84 days). Mice given intratracheal delivery of alloantigen plus anti-CD70, anti-CD134L, or anti-CD153 mAb, but not those given intratracheal delivery of alloantigen plus anti-CD137L mAb, rejected their graft acutely (MST 16, 14, 10, and 65 days, respectively). Adoptive transfer of splenocytes from mice pretreated with intratracheal delivery of alloantigen plus anti-CD70, CD134L, or CD153 mAb did not prolong survival of C57BL/10 cardiac grafts in naive secondary CBA recipients (MST 14, 11, and 11 days, respectively), whereas adoptive transfer of splenocytes from mice given intratracheal delivery of alloantigen plus anti-CD137L mAb did (MST 75 days). CONCLUSION: The CD27/CD70, CD134/CD134L, and CD30/CD153 pathways are independently required for generation of regulatory cells in our model.     

B7/CTLA4 pathway is essential for generating regulatory cells after intratracheal delivery of alloantigen in mice

BACKGROUND: The mechanism of hyporesponsiveness induced by intratracheal (IT) delivery of alloantigen was examined and its effect on cardiac graft survival was assessed in studies in mice. METHODS: In CBA (H2 ) mice, donor splenocytes were given by IT delivery 7 days before transplantation of a C57BL/10 (H2 ) heart. To determine whether regulatory cells were involved in hyporesponsiveness, splenocytes from mice given IT delivery of alloantigen and antibodies for B7-1, B7-2, or CTLA4 were adoptively transferred to na?ve secondary recipients 7 days after delivery; those recipients underwent heart transplantation the same day. Effects on cell proliferation and cytokine production of splenocytes from mice given IT delivery of alloantigen were examined in mixed leukocyte cultures (MLC). RESULTS: Cardiac graft survival was significantly prolonged in mice given IT delivery of alloantigen (median survival time [MST], 81 days); those given syngeneic splenocytes rejected grafts acutely (MST, 7 days; P<0.05). Adoptive transfer of splenocytes also significantly prolonged survival of cardiac grafts in secondary recipients (MST, 62 days). When B7-1, B7-2, or CTLA4 antibody was combined with IT delivery of alloantigen in the first recipient, all grafts were rejected within 14 days in second recipients after adoptive transfer. In mixed leukocyte cultures, splenocytes from these mice did not respond to alloantigen and production of interleukin-4 and interleukin-10 was increased. CONCLUSIONS: Donor splenocytes delivered IT induced hyporesponsiveness and regulatory cells in our animal model, and such induction was dependent on B7-1, B7-2, and CTLA4 signals.     

Nondepleting anti-CD4 monoclonal antibody enhances the ability of oral alloantigen delivery to induce indefinite survival of cardiac allografts: oral tolerance to alloantigen

BACKGROUND: We examined whether oral administration of alloantigen could induce the prolonged survival of cardiac allografts. METHODS: Hearts from CBK (H2k+Kb) transgenic or (C57BL/10xCBA)F1 (H2bxH2k) mice were transplanted into CBA (H2k) recipients pretreated orally with 1 x 10(7) donor splenocytes in the presence or absence of a nondepleting anti-CD4 (YTS 177, 200 microg/dose). RESULTS: Modest prolongation of CBK cardiac grafts was induced in CBA mice fed with multiple doses of CBK splenocytes (MST 42 days compared with controls fed with syngeneic CBA splenocytes, 12 days). When the CD4 monoclonal antibody, YTS177, was administered for 2 days before the first oral delivery of CBK splenocytes, all mice accepted their grafts indefinitely (MST > 100 days versus mice treated with anti-CD4 alone, 11.5 days). To determine if feeding multiple doses of alloantigen was essential, CBA mice were given CBK splenocytes orally on a single occasion in combination with the anti-CD4. The majority of the grafts survived indefinitely (MST >100 days). This oral treatment regimen also induced indefinite prolongation of (C57BL/10xCBA)F1 cardiac grafts. CONCLUSION: The induction of unresponsiveness by oral administration of alloantigen can be augmented by a nondepleting anti-CD4, YTS177, when given before the first oral delivery of allogeneic cells.     

Everolimus and mycophenolate mofetil are potent inhibitors of fibroblast proliferation after lung transplantation

BACKGROUND: Dysregulated fibroblast proliferation is thought to play an important role in the progression of bronchiolitis obliterans (BO) after lung transplantation. Augmented immunosuppression is often used to treat BO. We investigated the effect of methylprednisolone (mPRED), cyclosporine A (CsA), tacrolimus (FK506), azathioprine (AZA), mycophenolate mofetil (MMF), and everolimus (rapamycin derivative [RAD]) on the proliferative capacity of fibroblasts cultured from transbronchial biopsies of lung transplant recipients. METHODS: Primary cultures of human lung fibroblasts were obtained from 14 transbronchial biopsies of lung transplant recipients. Subconfluent cells were serum starved for 24 hr followed by growth stimulation in the presence or absence of the respective drug in six concentrations ranging as follows: 0.01 to 100 mg/L for mPRED; 0.01 to 50 mg/L for CsA and AZA; 0.001 to 5 mg/L for FK506 and MMF; and 0.00001 to 1 mg/L for RAD. Proliferation was quantified by [3H]thymidine incorporation and direct cell count. A toxic drug effect was excluded by trypan blue. RESULTS: Drug concentrations (mg/L) causing a 50% inhibition of fibroblast proliferation were mPRED 4; CsA 20; FK506 0.3; AZA 7; MMF 0.3; and RAD 0.0006. Drug concentrations (mg/L) causing inhibition of fetal bovine serum-induced proliferation were mPRED 60; CsA 45; FK506 3; AZA 35; MMF 1; and RAD 0.003. CONCLUSIONS: RAD and MMF were the most potent antifibroproliferative drugs and were effective at concentrations achieved clinically, supporting their use for the treatment of patients with early BO. Our method holds promise as an in vitro model to assess the likely in vivo responses of human lung fibroblasts to specific immunosuppressive drugs.     

Differential Effect of Diarrhea on FK506 Versus Cyclosporine A Trough Levels and Resultant Prevention of Allograft Rejection in Renal Transplant Recipients

Diarrhea is the most frequently reported adverse event in patients treated with mycophenolate mofetil. Twenty-six renal transplant patients on a mycophenolate mofetil-based immunosuppressive regime with persistent afebrile diarrhea were examined. Diarrhea caused a significant rise in FK-506 trough levels despite intake of stable doses, necessitating FK-506 dose reductions of 30% to obtain pre-diarrhea trough levels. In contrast, trough levels of cyclosporine A remained stable without dose adjustments. This suggests that absorption and/or metabolism is differentially altered for FK506 compared with cyclosporine A in patients with diarrhea. In nine patients mycophenolate mofetil was reduced or stopped because of persistent diarrhea without identifiable cause. This resulted in end-stage renal disease because of chronic rejection in two patients, and in acute rejection in two patients, all taking FK506 and steroids. Therefore, dose adjustments of FK506 in patients with diarrhea must be carefully monitored, especially when doses of mycophenolate mofetil are also reduced.     

Prolongation of skin allograft survival in rats by a novel immunosuppressive agent, FK506

FK506, an immunosuppresant, was isolated from Streptomyces tsukubaensis. Intramuscular administration of FK506 (0.32 mg/kg or more) 5 days a week for two weeks after grafting prolonged the acceptance time of F344 skin allograft to WKA rats. Similar results were obtained with cyclosporine at 32 mg/kg or more, but other immunosuppressives (i.e., prednisolone, azathioprine, and bredinin) gave only a marginal prolongation. The prolonging effect of FK506 was obtained in various donor-recipient combinations across a major or minor histocompatibility barrier. The agent also prolonged the acceptance time of mouse skin xenografts to rats. Furthermore, maintenance doses of 3.2 or 0.32 mg/kg twice a week after an initial 14-day treatment with the agent at 3.2 mg/kg gave graft survival as long as the treatment was continued for more than 120 days. Our findings show that FK506 has a potent immunosuppressive effect in rats and suggest that the agent merits further investigation.     

Effects of combination treatment with FK506 and cyclosporine on survival time and vascular changes in renal-allograft-recipient dogs

In our previous experiments studying the effects of FK506 on renal allografting in the dog, we encountered two major problems. One problem was anorexia and the other problem was vascular changes mainly in the recipient heart. Anorexia was generally dose dependent, but the vascular changes were seen to be more prominent at lower doses rather than at higher immunosuppressive doses. The present study was undertaken to study these two problems. A nonanorexic, vascular change-related, nonimmunosuppressive dose of FK506 was combined with a low dose of cyclosporine or prednisolone in beagle dogs after renal allografting. Treatment with either FK506 alone at a dose of 0.32 mg/kg or cyclosporine alone at 2.5 mg/kg was not effective in prolonging renal recipient survival. The recipient dogs died of rejection, and a variety of vascular changes were observed in the hearts of both groups. Combined treatment with FK506 and cyclosporine at these same doses resulted in statistically significant prolongation of the survival time of the renal recipient (P less than 0.01), and histologic studies showed that the frequency and severity of the vascular changes were suppressed in the recipient receiving the combined treatment. The combination of FK506 and prednisolone at 0.5 mg/kg was not effective in prolonging survival. Furthermore, the extent of vascular changes was similar to those found in recipients receiving FK506 alone. The data suggest that combined treatment with low doses of both FK506 and cyclosporine acted synergistically in prolonging canine renal allografts and that the vascular changes frequently seen at low doses of FK506 were reduced by additional immunosuppression with a low dose of cyclosporine.     

Induction of indefinite survival of fully mismatched cardiac allografts and generation of regulatory cells by sarpogrelate hydrochloride

BACKGROUND: At initiation of the immunologic response, platelets rapidly release chemical mediators such as serotonin (5-hydroxytryptamine, [5-HT]) and cytokines. Sarpogrelate hydrochloride (SH), a selective 5-HT2-receptor antagonist, is used to treat patients with peripheral arterial disease. We investigated the effect of SH on the alloimmune response in a murine cardiac transplantation model. METHODS: CBA mice underwent transplantation of a C57BL/10 heart and received a short course of SH treatment. Survival of the allograft was recorded. An adoptive transfer study was performed to determine whether regulatory cells were generated. Immunohistochemistry studies of intercellular adhesion molecule 1 (ICAM-1), histological, cell-proliferation, and cytokine assessments were performed. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 8 days). In mice given 10 mg/kg of SH, all allografts survived indefinitely (MST, >100 days); these mice also had significantly prolonged survival of donor-specific skin grafts but acute rejection of third-party skin grafts. Secondary CBA recipients given not only whole but also CD4 splenocytes from primary SH-treated CBA recipients with C57BL/10 cardiac allograft had indefinite survival of C57BL/10 hearts (MST, >100 days). SH inhibited upregulation of ICAM-1 on endothelial cells in the allografts. Graft acceptance and hyporesponsiveness were confirmed by the histological and cell-proliferation studies, respectively. Production of interleukin-4 and interleukin-10 from splenocytes of SH-treated transplant recipients increased compared to that from splenocytes of untreated recipients. CONCLUSION: SH induced indefinite survival of fully allogeneic cardiac allografts, generated CD4 regulatory cells, inhibited ICAM-1 expression in the allografts, and upregulated IL-4 and IL-10 production.     

Various costimulatory pathways are essential for induction of regulatory cells by intratracheal delivery of alloantigen

BACKGROUND: We previously reported that intratracheal delivery of alloantigen induced regulatory cells in a mouse heart transplantation model. We investigated the roles of costimulatory pathways in the induction of regulatory cells by intratracheal delivery of alloantigen. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of splenocytes (1 x 10(7)) from C57BL/10 (H-2b) mice and administration of monoclonal antibodies (mAb) specific for programmed death (PD)-1 and its ligands, programmed death-ligand (PD-L)1 and PD-L2, CD70, CD134 ligand (CD134L), CD153, CD137L, or receptor activator of nuclear factor-kappaB (NF-kappaB) (RANK). Seven days later, naive CBA mice underwent adoptive transfer of splenocytes (5 x 10(7)) from the pretreated CBA mice and transplantation of C57BL/10 heart. RESULTS: Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST], 68 days) as compared with adoptive transfer from untreated CBA mice (MST, 12 days). Concomitant administration of control immunoglobulin (Ig)G, anti-PD-L2 mAb, or anti-CD137L along with intratracheal delivery did not significantly affect the prolongation (MST, 72, 68, and 65 days, respectively). In contrast, anti-PD-1, anti-PD-L1, anti-CD70, anti-CD134L, anti-CD153, or anti-RANK mAb abrogated the prolongation induced by adoptive transfer from the pretreated mice with intratracheal delivery (MST, 18, 17, 16, 14, 10, and 18 days, respectively). CONCLUSION: The PD-1/PD-L1, CD27/CD70, CD134/CD134L, CD30/CD153, and tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE)/RANK interactions are independently required for generation of regulatory cells by intratracheal delivery of alloantigen.     

FK506 as the sole immunosuppressive agent for prolongation of islet allograft survival in the rat.

The purpose of the present study was to determine immunosuppressive effects of a new immunosuppressive agent, FK506, on rat islet allografts and also whether FK is toxic to the islet grafts since the diabetogenic effects of FK is controversial. Hand-picked clean fresh islets (WKA/Qdj:RT1u) were transplanted either beneath the renal capsule or into the liver via the portal vein of the diabetic (STZ, 60 mg/kg) rats (Lewis:RT1(1)). FK506 was administered s.c. for 7 days after transplantation. The mean survival times (MST)* of the renal subcapsular grafts receiving 0 (control), 0.32 or 1.0 mg/kg FK were 7.2 +/- 1.1 (mean +/- SD, n = 5), 13.8 +/- 4.8 (n = 4), and 20.2 +/- 8.0 days (n = 5), respectively. The MST of the intrahepatic grafts receiving 0, 0.1, 0.32, or 1.0 mg/kg FK were 4.4 +/- 1.1 (n = 5), 7.2 +/- 0.8 (n = 5), greater than 45.3 +/- 23.1 (n = 6) or greater than 54.4 +/- 8.8 days (n = 5), respectively. Histologically, islets were found easily in the liver of normoglycemic recipients for more than 60 days after transplantation and appeared intact, with well-granulated beta cells. Foci of mononuclear cells were occasionally seen adjacent to the islet cells. The plasma glucose of the recipients with 1.0 mg/kg FK fluctuated between 150 and 350 mg/dl without rejection. In the recipients treated with 3.2 mg/kg FK the plasma glucose of all the recipients (n = 3) returned to pretransplant levels by 21 days after transplantation. However, islet cells were present in the liver of all these recipients without mononuclear cell infiltration. Immunohistochemically islet grafts stained weakly for insulin, but to the same extent as the controls for glucagon and somatostatin. These findings clearly demonstrate the immunosuppressive effect of FK506 on islet allografts and the importance of the transplant site for prolongation of graft survival by FK, and also suggest that FK has toxic effects on the islet grafts (B cells) when used in high dosages.     

Danazol induces prolonged survival of fully allogeneic cardiac grafts and maintains the generation of regulatory CD4(+) cells in mice

Danazol, a derivative of testosterone, is useful for treatment of endometriosis as well as pretreatment for in vitro fertilization and embryo transfer, although its mechanisms of action are unclear. The aim of this study was to investigate the effect of danazol on alloimmune responses in murine heart transplantation. CBA male mice (H2(k) ) underwent transplantation of C57BL/6 male (H2(b) ) hearts and received a single dose of danazol (0.4, 1.2 or 4mg/kg/day) by intraperitoneal injection on the day of transplantation and for 6days thereafter. An adoptive transfer study was performed to determine whether regulatory cells were generated. The median survival time (MST) of allografts in danazol-treated (1.2 and 4mg/kg/day) mice was 28 and 63days, respectively, compared with 7days in untreated mice. Moreover, secondary CBA recipients given whole splenocytes or CD4(+) cells from primary danazol-treated (4mg/kg/day) CBA recipients 30days after transplantation had prolonged allograft survival (MSTs, 29 and 60days, respectively). Cell proliferation, interleukin (IL)-2 and interferon-γ were suppressed in danazol-treated mice, whereas IL-4 and IL-10 were up-regulated. Moreover, danazol directly suppressed allo-proliferation in a mixed leukocyte culture. Flow cytometry showed an increased CD4(+) CD25(+) Foxp3(+) cell population in splenocytes from danazol-treated mice. Danazol prolongs cardiac allograft survival and generates regulatory CD4(+) cells.     

Immunophilin ligands FK506 and cyclosporine A improve neurologic and histopathologic outcome after transient spinal cord ischemia in rabbits

BACKGROUND: We comparatively evaluated the protective effect of the immunophilin ligands cyclosporine A (INN: ciclosporin), FK506, and rapamycin on the spinal cord in a rabbit model of transient ischemia. Both cyclosporine A and FK506 inhibit calcineurin, whereas rapamycin does not. METHODS: Thirty-six male New Zealand White rabbits were divided into the following 6 groups: group C, 15 minutes of spinal cord ischemia; group FK, FK506 (1 mg/kg) administered 30 minutes before ischemia; group CsA, cyclosporine A (30 mg/kg) administered 30 minutes before ischemia; group CsA-C, chronic administration of cyclosporine A (20 mg/kg) for 9 days before ischemia; group R, rapamycin (1 mg/kg) administered 30 minutes before ischemia; and group R+FK, rapamycin (1 mg/kg) administered 20 minutes before FK506 pretreatment (1 mg/kg). Group CsA-C was added because the drug does not readily cross the blood-brain barrier. Neurologic function was evaluated by Johnson's 5-point scale at 8, 24, and 48 hours after ischemia, and histopathology was assessed 48 hours after ischemia. RESULTS: At 24 and 48 hours after ischemia, the Johnson score was better in groups FK (4.0 +/- 1.1), R+FK (3 +/- 1.1), and CsA-C (2.7 +/- 1.2) than in group C (0.8 +/- 1.2). Numbers of morphologically intact anterior horn cells were higher in groups FK (31.3 +/- 9.9), R+FK (23.2 +/- 4.5), and CsA-C (18.3 +/- 6.8) than in group C (6.3 +/- 4.3). CONCLUSIONS: FK506 and chronic administration of cyclosporine A, but not rapamycin, protect the spinal cord from transient ischemia. Although these results are compatible with inhibition of calcineurin in the mechanism of neuroprotective action of these drugs, other effects through different pathways cannot be excluded before further study.     

Synergistic effect of vincristine with tacrolimus or sirolimus in prevention of acute heart allograft rejection in the rat

The application of multiple immunosuppressive therapy for organ transplantation could enhance therapeutic efficacy, while minimizing the toxicity of individual drugs used in the regimen. In this study, the effect of the combined therapy of vincristine (VCR) with tacrolimus (FK506) or sirolimus (rapamycin, RAPA) was tested in prevention of acute heart allograft rejection in the rat. A Brown Norway (BN, RT 1(n)) to Lewis (LEW, RT 1(1)) rat combination was used in a heart allografting model. VCR was administered intraperitoneally once daily, while FK506 and RAPA were given by gavage once daily for 14 days after transplantation. There were dose-related prolongations of mean survival time (MST) to monotherapy of VCR, FK506, or RAPA. The MST in combination therapy indicated that a synergistic interaction was produced when compared with the respective monotherapies: VCR 0.05 mg/kg/day + FK506 0.5 mg/kg/day (16.00 +/- 1.79 days, P = 0.001; combination index (CI) = 0.557); VCR 0.05 mg/kg/day + FK506 1.0 mg/kg/day (29.00 +/- 10.54 days, P = 0.001; CI = 0.598); VCR 0.05 mg/kg/day + RAPA 0.2 mg/kg/day (17.33 +/- 1.97 days, P = 0.001; CI = 0.500); and VCR 0.05 mg/kg/day + RAPA 0.4 mg/kg/day (21.17 +/- 3.19 days, P = 0.001; CI = 0.838). Combination therapy of VCR and FK506 or RAPA produced synergistic effects in prevention of acute heart allograft rejection in the rat.     

The smell of Tokishakuyaku-san (TJ-23) induces generation of regulatory T cells and prolongation of survival of fully allogeneic cardiac grafts in mice

Oral administration of Tokishakuyaku-san (TJ-23), a Japanese herbal medicine, induces prolongation of cardiac allograft survival and generates regulatory cells in mice. Because herbal medicines usually have unique odor, and because smell is supposed to modulate the immune system, we examined whether the odor of TJ-23 induced prolonged allograft survival and regulatory cell generation. Naïve CBA mice (H2^k) and olfactory-dysfunctional CBA mice after a stereotaxic operation underwent transplantation of C57BL/6 (B6, H2^b) hearts, receiving fumigated water only or TJ-23 until rejection. Untreated or treated with water fumigation CBA mice rejected B6 cardiac grafts acutely (median survival times [MSTs], 7 and 8.5 days). When CBA mice were treated with fumigation of TJ-23, allograft survival was significantly prolonged (MST, 48 days). Olfactory-dysfunctional CBA mice treated with fumigation of TJ-23 rejected grafts acutely (MST, 7 days). Treatment with fumigation of TJ-23 also suppressed splenocytes proliferation and interferon-γ production. Secondary CBA recipients of whole splenocytes or CD4⁺ cells from primary TJ-23-treated CBA recipients of B6 cardiac allografts at 30 days after grafting showed prolonged survival of B6 hearts (MST, >60 days). Flow cytometry studies showed increased CD4⁺CD25⁺Foxp3⁺ regulatory cells in recipients given fumigation of TJ-23. In conclusion naïve but not olfactory-dysfunctional CBA mice treated with fumigation of TJ-23 displayed prolonged survival of fully allogeneic cardiac allografts and generation of regulatory cells.